Split me baby one more time!

Only a short post but hey, bloggers gonna blog! Also please try to appreciate the pun title by the end of this blog, I’m so happy I came up with it!

So I am now about two thirds of the way through my biology project and it has been a hard knock life getting into the lab for 11am and pipetting various liquids and solutions together to produce mutated versions of the GTPase DEF6. My lab partner and I are currently trying to images of the mutated DEF6 plasmid with varying levels of success. The cells we are using are called COS-7 cells which produce actin filaments similar to T-cells when they are activated by pathogens in the blood.

A continuing task throughout this project has been to sustain cultures of the COS-7 cells, making sure that there is a dense enough sample of them, readily available for us to transfect (inserting plasmids) the cells with the mutated plasmid. The cells are kept in a standard petri dish with cell culture media containing 10% Foetal Bovine Serum (FBS). The FBS is a supplemental serum containing growth factors but with little antibodies and is a standard addition to a medium for the culturing of eukaryotic cells. As the cells divide rapidly and quickly form a dense population, the media needs to be removed and a fraction of the cells are transferred to a new petri dish. This prevents the media from being depleted of nutrients and the cells from becoming saturated, both of which would lead to the cells dying and me being unhappy. This is called splitting cells (or at least thats what I call it)

As the cells need to be kept uncontaminated by any cells such as bacteria, the entire method of splitting the cells into a subculture is carried out in a fume cupboard safety cabinet which extracts air from the cabinet and replaces with clean air to ensure a sterile environment. The first step in this ever-exciting process (I’m a tad sarcastic) is to remove the current media in the petri using a 10ml pipette and discard in disinfectant. Don’t worry no cells are removed in this process because COS-7 cells are adherent – stick to the surface – and will remain in the petri dish. The residual media remaining needs to be washed out with a buffer solution – phosphate-buffered saline (PBS) is used due to being non-toxic and having similar ion concentration to the COS-7 cells used. Adding 5ml of PBS to the dish is enough to wash the cells and helps to reduce the likelihood of contamination.

My cosy little safety cabinet where I split my cells

As the cells are adherent, they need to be ‘unstuck’ from the plate in order to be divided using trypsin – a protease found in the digestive system – which breaks down the proteins allowing the cells to stick to the dish surface. A small amount of trypsin is added, removed shortly after and incubated for a few minutes so the residual trypsin can act upon the cell’s adhesion proteins. To ensure the trypsin has done its job on most cells, the petri dish is lightly tapped a few times and observed through a microscope where it should be seen that the cells are moving around – it looks like in the picture below.

Now the cells are free to be split into a subculture! Adding some fresh new medium with 10% FBS to a new petri dish and a small amount of the original cell-containing medium is all that’s needed for this new culture to be made. The new culture is then incubated until around two days’ time when they’ll need to be split again. The original culture is thrown away and cleaned out with disinfectant.

And there we have the method of splitting cells into a subculture. Its massively exciting and I bet you can’t wait to go split your very own COS-7 cell line culture for transfection!